U.S. patent number 5,798,263 [Application Number 08/708,533] was granted by the patent office on 1998-08-25 for apparatus for quantifying dual-luminescent reporter assays.
This patent grant is currently assigned to Promega Corporation. Invention is credited to Bruce A. Sherf, Keith V. Wood.
United States Patent |
5,798,263 |
Wood , et al. |
August 25, 1998 |
Apparatus for quantifying dual-luminescent reporter assays
Abstract
The presently disclosed invention is drawn to an automatic
luminometer apparatus capable of measuring two distinct luminescent
reactions from within a single, non-compartmentalized sample
container. The present apparatus may be dimensioned, configured,
and programmed to automatically perform dual-reporter luminescent
assays using multi-well sample plates, such as 96-well microtiter
plates.
Inventors: |
Wood; Keith V. (Madison,
WI), Sherf; Bruce A. (Waunakee, WI) |
Assignee: |
Promega Corporation (Madison,
WI)
|
Family
ID: |
24846167 |
Appl.
No.: |
08/708,533 |
Filed: |
September 5, 1996 |
Current U.S.
Class: |
435/288.7;
250/361C; 356/246; 422/52; 422/82.05; 435/287.2; 435/288.4 |
Current CPC
Class: |
G01N
21/763 (20130101) |
Current International
Class: |
G01N
21/76 (20060101); C12M 001/34 () |
Field of
Search: |
;435/6,8,288.3,288.4,288.7,287.3,287.2,808 ;422/52,65,82.05,82.08
;436/172,805 ;250/361C ;356/246 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Other References
Blaise, C., et al. (1994) BioTechniques: 16, 932-937. .
Bronstein, I., et al., (1994) Anal-Biochem.: 219, 169-181. .
Denburg, et al. (1969) Archives of Biochemistry and Biophysics:
134, 381-394. .
Denburg, J.L., and McElroy, W.D. (1970) Archives of Biochemistry
and Biophysics: 141, 668-675. .
Flanagan, W. M. et al. (1991) J. Virology: 65, 769-786. .
Jain, V. K. and Magrath, I. T. (1992) BioTechniques: 12, 681-683.
.
Kondepudi, T., et al., Poster abstract #725, presented at annual
meeting of the American Society of Cell Biology, Dec. 10-14, 1994,
San Francisco, CA. .
Leckie, F. et al. (1994) BioTechniques: 17, 52-57. .
Lee et al. (1970) Archives of Biochemistry and Biophysics: 141,
38-52. .
Thompson, J. F., et al. (1991) Gene: 103, 171-177..
|
Primary Examiner: Beisner; William H.
Attorney, Agent or Firm: DeWitt Ross & Stevens SC
Claims
What is claimed is:
1. An apparatus for quantifying integrated photon-generating assays
which utilize two distinct photon-generating reactions within a
non-compartmentalized sample container, the apparatus
comprising:
sample holding means dimensioned and configured to releasibly grip
a non-compartmentalized sample container workpiece;
injecting means for sequentially injecting distinct first and then
second reagents into the sample container workpiece at a
user-defined time interval greater than zero;
a single photon measuring means for measuring photons emanating
from the sample container workpiece and generating signals
proportional to the measured photons, the photon measuring means
dimensioned and configured to sequentially generate a first
corresponding signal subsequent to injection of the first reagent
into the sample container workpiece and a second corresponding
signal subsequent to injection of the second reagent into the
sample container workpiece;
signal storage and retrieval means for storing and retrieving the
first and second signals, the signal storage and retrieval means
operationally linked to the photon measuring means;
programmable control means operationally linked to and dimensioned
and configured to control operation of the injecting means, the
photon measuring means, and the signal storage and retrieval means,
the programmable control means further dimensioned and configured
to perform mathematical manipulations on the first and second
signals generated by the photon measuring means; and
display means to display the first and second signals and any
mathematical manipulations performed thereon.
2. The apparatus according to claim 1, wherein the sample holding
means is dimensioned and configured to releasibly grip a single,
non-compartmentalized sample cuvette.
3. The apparatus according to claim 1, wherein the sample holding
means is dimensioned and configured to releasibly grip a 96-well
microtiter plate in which individual wells of the microtiter plate
are non-compartmentalized.
4. The apparatus according to claim 1, wherein the injecting means
comprises cooperating first and second injecting means for
sequentially injecting the distinct first and then second reagents,
respectively, into the sample container workpiece.
5. The apparatus according to claim 1, wherein the injecting means
comprises at least two reservoirs to contain the first and second
reagents, respectively, and at least two corresponding delivery
means for delivering the first and second reagents from the at
least two reservoirs to the sample container workpiece.
6. The apparatus according to claim 5, wherein the delivery means
is selected from the group consisting of peristaltic pumps,
reciprocating pumps, and vacuum aspirators.
7. The apparatus according to claim 1, wherein the photon measuring
means is selected from the group consisting of single
photomultiplier tubes, compound photomultiplier tubes, avalanche
diodes, and charge-coupled devices.
8. The apparatus according to claim 1, wherein the programmable
control means is one or more microprocessor control means.
9. The apparatus according to claim 1, wherein the injecting means
includes an outflow orifice disposed adjacent to the sample
container workpiece, and wherein the outflow orifice, the sample
holding means, and the photon measuring means are disposed within a
light-tight enclosure.
10. The apparatus according to claim 1, further comprising means
for quantifying a baseline signal of the photon measuring
means.
11. An apparatus for quantifying integrated photon-generating
assays which utilize two distinct photon-generating reactions
within a non-compartmentalized sample container, the apparatus
comprising:
sample holding means dimensioned and configured to releasibly grip
a non-compartmentalized sample container workpiece;
first injecting means for injecting a first reagent into the sample
container workpiece;
photon measuring means for measuring photons emanating from the
sample container workpiece at a user-defined time interval
subsequent to injection of the first reagent and for generating a
signal proportional to the measured photons;
second injecting means for injecting a second reagent into the
sample container workpiece at a user-defined time interval greater
than zero subsequent to injection of the first reagent;
photon measuring means for measuring photons emanating from the
sample container workpiece at a user-defined time interval
subsequent to injection of the second reagent and for generating a
signal proportional to the measured photons, wherein the photon
measuring means for measuring photons emanating from the sample
container workpiece at a user-defined time interval subsequent to
injection of the first reagent and the photon measuring means for
measuring photons emanating from the sample container workpiece at
a user-defined time interval subsequent to injection of the second
reagent are one in the same;
signal storage and retrieval means for storing and retrieving the
first and second signals, the signal storage and retrieval means
operationally linked to the photon measuring means;
programmable control means operationally linked to and dimensioned
and configured to control operation of the first and second
injecting means, the photon measuring means, and the signal storage
and retrieval means, the programmable control means further
dimensioned and configured to perform mathematical manipulations on
the first and second signals generated by the photon measuring
means; and
display means to display the first and second signals and any
mathematical manipulations performed thereon.
12. The apparatus according to claim 11, wherein the programmable
control means is one or more microprocessor control means.
13. The apparatus according to claim 11, wherein the injecting
means includes one or more outflow orifices disposed adjacent to
the sample container workpiece, and wherein the one or more outflow
orifices, the sample holding means, and the photon measuring means
are disposed within a light-tight enclosure.
Description
This applilcation claims the benefit of U.S. Privisional
Application No. 60/003,284, filed Sep. 5, 1995.
FIELD OF THE INVENTION
The present invention is drawn to an apparatus for quantifying
integrated, single-tube, dual-reporter luminescent assays.
Specifically, the present invention relates to an apparatus which
allows two distinct luminescent reactions to be assayed
sequentially within a single, non-compartmentalized sample.
CITED REFERENCES
Full bibliographic citations to the references cited in this
provisional application can be found in the Bibliography
section.
DESCRIPTION OF THE PRIOR ART
Luminescence is produced in certain organisms as a result of
luciferase-mediated oxidation reactions. Currently, luciferase
genes from a wide variety of vastly different species, particularly
the luciferase genes of Photinus pyralis (the common firefly of
North America), Pyrophorus plagiophathalamus (the Jamaican click
beetle), Renilla reniformis (the sea pansy), and several bacteria
(e.g., Xenorhabdus luminescens and Vibrio spp), are extremely
popular luminescence reporter genes. Reference is made to
Bronstein, et al. (1994) for a review of luminescence reporter gene
assays. Firefly luciferase is also a popular reporter for ATP
concentrations, and in that role is widely used to detect biomass.
Various other reporter applications of luciferases have been
described in the scientific literature. Luminescence may be
produced by other enzymes when mixed with certain synthetic
substrates; such as alkaline phosphatase mixed with adamantyl
dioxetanes, or horseradish peroxidase mixed with luminol.
Luciferase genes are widely used as genetic reporters due to the
non-radioactive nature, sensitivity, and extreme linear range of
luminescence assays. For instance, as few as 10.sup.-20 moles of
the firefly luciferase can be detected. Consequently, luciferase
assays of gene activity are used in virtually every experimental
biological system, including both prokaryotic and eukaryotic cell
cultures, transgenic plants and animals, and cell-free expression
systems. Similarly, luciferase assays of ATP are highly sensitive,
enabling detection to below 10.sup.-16 moles.
Luciferases generate light via the oxidation of enzyme-specific
substrates, called luciferins. For firefly luciferase and all other
beetle luciferases, this is done in the presence of magnesium ions,
oxygen, and ATP. For anthozoan luciferases, including Renilla
luciferase, only oxygen is required along with the luciferin.
Generally, in luminescence assays of genetic activity, reaction
substrates and other luminescence-activating reagents are
introduced into a biological system suspected of expressing a
reporter enzyme. Resultant luminescence, if any, is then measured
using a luminometer or any suitable radiant energy-measuring
device. The assay is very rapid and sensitive, and provides gene
expression data quickly and easily, without the need for
radioactive reagents. Reporter assays other than for genetic
activity are performed analogously.
The conventional assay of genetic activity using firefly luciferase
has been further improved by including coenzyme A (CoA) in the
assay reagent to yield greater enzyme turnover and thus greater
luminescence intensity. (Promega Luciferase Assay Reagent, Cat. No.
E1500, Promega Corporation, Madison, Wis.; see U.S. Pat. No.
5,283,179, incorporated herein by reference.) Using this reagent,
luciferase activity can be readily measured in luminometers or
scintillation counters. The luciferase reaction, modified by the
addition of CoA to produce persistent light emission, provides an
extremely sensitive and rapid assay for quantifying luciferase
expression in genetically altered cells or tissues.
The concept of a dual-enzyme reporter system relates to the
simultaneous use and measurement of two individual reporter enzymes
within a single system. In genetic reporting, examples that
currently benefit from dual-reporter assays include individual
cells or cell populations (such as cells dispersed in culture,
segregated tissues, or whole animals) genetically manipulated to
simultaneously express two different reporter genes. Most
frequently, the activity of one gene reports the impact of the
specific experimental conditions, while the activity of the second
reporter gene provides an internal control by which all sets of
experimental values can be normalized.
Cell-free reconstituted systems that may benefit from dual-enzyme
reporter technology are cellular lysates derived for the
simultaneous translation, or coupled transcription and translation,
of independent genetic materials encoding experimental and control
reporter enzymes. Immuno-assays may, likewise, be designed for
dual-reporting of both experimental and control values from within
a single sample.
Currently, genes encoding firefly luciferase (luc), chloramphenicol
acetyl transferase (CAT), beta-galactosidase (lacZ),
beta-glucuronidase (GUS) and various phosphatases such as secreted
alkaline phosphatase (SEAP) and uteroferrin (Uf; an acid
phosphatase) have been combined and used as co-reporters of genetic
activity. The following references provide representative examples
of these various reporter genes used in combined form for the
purpose of dual-reporting of genetic activity: luc and GUS: Leckie
et al. (1994); luc and CAT, and luc and lacZ: Jain and Magrath
(1992); CAT and lacZ: Flanagan et al., (1991); SEAP and Uf:
Kondepudi et al., (1994).
The performance of any dual-enzyme reporter assay is limited by the
characteristics of the constituent enzyme chemistries and the
ability to correlate their respective resulting data sets.
Quantifying the combined expression of any two of the above
reporters from within a single cell lysate necessitates splitting
the sample so that the activity of each reporter can be assayed
independently. Hence, the disparate enzyme kinetics, assay
chemistries and incubation requirements, of these various reporter
enzymes makes it impossible to combine them into an integrated,
single-tube, dualreporter assay format. An ideal dual-reporter
system would be comprised of two enzyme assays with compatible
chemistries, and identical temperature and handling conditions,
speed, sensitivity, and instrumentation required for detection.
For instance, U.S. Pat. Nos. 5,035,866 and 5,159,197, to Wannlund,
describe a relatively simple apparatus for performing
bioluminescent bacteriuria analysis. The analysis is based upon a
measurement of bacterial ATP from any bacteria present in the urine
sample. Before the luminescent reaction can be initiated,
non-bacterial ATP must be removed from the sample so it will not
result in a false positive reading. Wannlund utilizes multi-well
test plates having corresponding upper and lower chambers. The
urine sample is placed into the upper chamber and the chemicals
necessary to remove non-bacterial ATP are added to the sample.
After this reaction is completed, the sample is forced into the
lower chamber by air pressure, where the reagents necessary to
initiate the luminescent reaction are already in place. The sample
plate is then mechanically biased against a conventional high-speed
photographic film plate. Those samples testing positive for
bacterial infection will emit a luminescent signal, thereby
exposing the photographic film directly beneath that test well. The
apparatus is qualitative, simple in operation, and does not require
an external power source. This device, however, is solely
qualitative in nature, and is incapable of rendering meaningful
quantitative distinctions between luminescent samples.
U.S. Pat. No. 4,818,883 to Anderson et al. describes a luminometer
apparatus in which a shutter is situated between the sample and a
photo-detector. Light produced during a phosphorescent reaction is
detected by the photo-detector and signals output from the
photo-detector are applied to a circuit which samples the signals
from the photo-detector at preset intervals. The successive values
of the signals are subtracted from one another to determine a peak
value of light intensity. Once the peak value of light intensity
has been determined the shutter is interposed between the sample
and the photo-detector and the dark current signal of the
photo-detector is measured. The dark current signal is then
subtracted from the peak measured value of the phosphorescent
reaction. The true peak intensity of the emitted light from the
phosphorescent reaction is thereby determined. To determine the
concentration of the material being assayed in the sample, the peak
intensity is raised to a given exponent, which value has been
previously derived for the analyte being sampled. This arrangement
of elements is similar to many common luminometers where the dark
current of the photo-detector is determined, followed by
acquisition of the sample signal, and subtraction of the dark
current from the sample signal to determine the true luminescent
intensity of the sample.
U.S. Pat. No. 5,290,708 to Ashihara et al. describes an apparatus
for automatically performing immunoassays. The apparatus utilizes
bifurcated cartridges having two separate components separated by a
sealing film. The reactants for the immunoassay being performed are
separately contained within the two compartments. The sealing film
between the two compartments is broken to initiate the immuno
reaction under study. The apparatus includes means to convey the
cartridges along a reaction line where the immuno reaction is
incubated and ultimately measured. The reaction line is steppingly
movable and includes stations for diluting, stirring, washing,
aspirating, and measuring the immuno reaction. The entire apparatus
is controlled by a device having a memory storing operator which is
capable of selecting programs for various measuring methods.
A similar automated immunoassay analyzer is described in U.S. Pat.
No. 5,316,726 to Babson et al. This patent discloses a
computer-controlled instrument capable of performing a wide variety
of immunoassays and providing real-time presentation of the
operations being performed by the instrument. The instrument is
capable of performing more than one type of immunoassay on any one
given sample. The movement of the immunoassay sample tubes through
the apparatus is controlled by a bar code reader and a computer
controller. The apparatus uses assay tubes which allow water to be
expelled by centrifugal force, rather than by aspiration. This
apparatus can measure immuno reactions using fluorescent,
radioactive, or chemiluminescent labels.
Another automatic chemical analyzing device is described in U.S.
Pat. No. 5,380,487 to Choperena et al. This computer-controlled
device is an analyzer which permits automatic analysis of samples
for multiple analytes using different assay protocols in a multiple
chronology sequence, while operating on a predetermined and fixed
length cycle. Here, certain standard steps, such as incubation,
washing, and signal detection are assigned fixed operating
sequences which begin and end within a fixed timing cycle. The
fixed time duration allows samples to be transferred directly from
one assay station to another without unnecessarily occupying any
unused stations. In effect, each sample cuvette and each station
within the apparatus are assigned a fixed time slot. This
simplifies maximizing the throughput of the apparatus while
minimizing the complexity of the logic and control of the various
assay stations.
European Patent Application 0 025 350 to Holley describes an
apparatus for detecting luminescent reactions in multi-well plates.
The apparatus includes a plurality of liquid injector tubes
situated above the multi-well plate and a corresponding plurality
of photo-detectors situated below the multi-well plate. An entire
row of sample wells are positioned in registration with both the
liquid injector tubes and the photo-detectors. Reagents are then
injected into the sample wells to initiate a luminescent reaction
within each sample well. The luminescence generated by these
reactions is then measured by the photo-detectors. The entire
process is then repeated by positioning the next row of sample
wells in registration with the injector tubes and photo-detectors.
This reference does not comprehend an apparatus in which more than
one reagent, or more than one distinct luminescent reaction is
assayed within each sample.
Related devices for automatically analyzing luminescent reactions
within a plurality of separate sample cuvettes are described in
U.S. Pat. No. 3,756,920 to Kelbaugh et al., U.S. Pat. No. 4,459,265
to Berglund, and U.S. Pat. No. 4,755,055 to Johnson et al. These
references all describe various arrangements for transporting a
plurality of sample cuvettes into operational relationship with an
injector and photomultiplier tube for initiating and quantifying a
luminescent reaction within the sample. Like the above references,
none of these references described performing a dual-luminescent
assay within a single sample.
U.S. Pat. No. 5,043,141 to Wilson et al., U.S. Pat. No. 5,082,628
to Andreotti et al., and U.S. Pat. No. 5,139,745 to Barr et al. all
describe luminometer devices having an injector apparatus situated
above a sample to be tested, and a photo-detector situated below
the sample. The injector adds the necessary reagents to initiate a
luminescent reaction, whose energy is then detected by the
photo-detector.
A major drawback to the use of luminescence assays in
high-throughput applications is their incompatibility with standard
laboratory equipment. For instance, it is not possible to quantify
luminescence reactions contained in clear multi-well plates with
precision or accuracy because of the internal refraction of light
through the optically clear plate. FIG. 1 demonstrates that, when
using conventional 96-well clear polystyrene microtiter plates, the
luminescence signal generated in one well is refracted through the
plastic over relatively long distances. Hence, the light refracted
from one luminous sample can interfere with the subsequent
measurement of signal from luminescent samples in successive wells.
FIG. 2 shows the cumulative nature of refracted light emanating
from multiple luminous samples within a single clear plastic plate.
While the luminescent signal in the first sample well can be
measured accurately, sequential activation of luminescent reactions
in following wells leads to increasingly inaccurate measurements
due to the cumulative emission of photons refracted through the
plastic from all previous samples. This problem of refracted light,
or "refractive cross-talk" is further exacerbated when brightly
illuminated wells are situated adjacent to negative control wells
in which no luminescence is generated, or when brightly lit wells
are situated near relatively dim wells. This makes determining the
absolute and baseline luminescence in a clear multi-well plate
difficult, if not impossible.
Multi-well plates made from opaque plastics such as white and black
polyethylene are commercially available (e.g., DynaTech
Laboratories, Chantilly, Va.; Labsystems, Helsinki, Finland; NUNC,
Roskilde, Denmark), and are now being adopted to prevent refractive
cross-talk between samples in applications involving
high-throughput luminometric analysis (Blaise, C., et al., 1994).
However, while the reflectivity of white plastic yields greater
luminescence sensitivity than clear plates, photons are readily
scattered from the walls of adjacent wells, again introducing
cross-talk interference between wells. Here, the cross-talk is
referred to as "reflective cross-talk." In the same manner as
refractive cross-talk, reflective cross-talk is particularly
evident when assaying dim wells (such as negative controls not
containing luciferase) that are adjacent to bright wells. Black
96-well plates, originally intended for fluorescent applications,
are not ideal for luminescence applications because the sample
signal is greatly diminished due to the non-reflective nature of
the plastic.
Regardless of color, the cost of opaque plates as compared to
conventional transparent plates is substantial. Opaque plates
currently cost approximately $5 to $6 each, as compared to
transparent plates which normally retail for less than half that
amount. For example, sterile opaque 96-well plates and lids are
offered by DynaTech Laboratories at a combined retail cost of $286
per set of 50, or $5.72 per each. Similarly treated transparent
96-well plates with lids are manufactured by Corning (Corning,
N.Y.) and can be purchased at a retail cost of $110 per 50, or
$2.20 per each.
In addition to their cost, opaque plates impose technical
limitations not associated with clear multi-well plates. For
example, many researchers desire to expedite their assay operations
and reduce the cost of materials by culturing cells directly in the
wells of the microplate used to perform the final assay. Opaque
plates are inferior for this purpose because:
i) cultured cells cannot be viewed or photographed through the
opaque plate;
ii) the composition and surface characteristics of opaque plastics
are different from those of standard cell culture-grade
plastic-ware, and have undetermined effects on cell adhesion and
growth characteristics; and
iii) sterile, cell-culture grade opaque plates and covers (packaged
separately) are not widely available.
One manufacture (Packard, Meridian, CT) recognized the
technological problems associated with opaque plates and responded
by developing a specialty plate consisting of an opaque plastic
body with a clear plastic bottom (sold under the name "View Plate",
product #600-5181). However, the availability of such specialty
plates is limited, and the high price ($6 each) of such a
consumable product is generally prohibitive for highthroughput
users in both academic and private laboratories.
The 1991 publication of Thompson et al. presents findings on the
use of substrate analogs (benzothiazole, phenylbenzothiazole, and
hydroxy-phenylbenzothiazole) to induce conformational changes in
firefly luciferase. Thompson et al. demonstrate that, when bound to
the luciferase enzyme, said chemical compounds provide increased in
vivo and in vitro stability to the luciferase enzyme by conferring
greater resistance to proteolytic degradation. Though analogs to
beetle luciferin are inhibitors of firefly luciferase activity, the
assay of luciferase activity from treated samples was performed
using diluted cellular extracts containing sub-inhibitory
concentrations of the various residual substrate analogs.
U.S. Pat. No. 4,235,961 to A.T. Lundin describes a method for the
photometric determination of subunit B of creatinine kinase. The
assay proceeds in the presence of the L-luciferin enantiomer of the
natural beetle luciferase substrate (D-luciferin), which acts as a
competitive inhibitor of the luciferase/D-luciferin reaction.
Inhibition of the photometric reaction provides a more continuous
emission of light from the sample, thereby allowing the kinetics of
creatine kinase reaction to be studied.
U.S. Pat. No. 4,390,274 to Berthold et al. describes a photometric
assay in which an additional luminescence substrate is added to a
sample after a first experimental photometric measurement is taken.
A second photometric measurement is then taken. The added substrate
is chemically distinct from the experimental substrate being
measured, but is a reaction partner in the same luminescence
reaction system that is photometrically determined in the first
measurement. The added substrate is used for internal
standardization of each sample.
Various other publications describe chemical compounds which will
reduce the luminescence of a luciferase reaction, but in none of
these is this reduction of purposeful value in itself. Denburg and
McElroy (1970) present findings on the interaction of selected
anions as inhibitors of the firefly luciferase luminescent
reaction. Thiocyanate, iodide, nitrate, bromide and chloride are
found to have varying inhibitory interaction with the luciferase
enzyme. Lee et al. (1970) and Denburg et al. (1969) present
findings on various competitive inhibitors of the firefly
luciferase luminescent reaction.
None of the above references, taken alone, or in any combination,
are seen as describing the presently disclosed invention.
SUMMARY OF THE INVENTION
A first embodiment of the present invention is an apparatus for
quantifying integrated photon-generating assays. These assays
utilize two distinct photon-generating reactions within a
non-compartmentalized sample container. The apparatus comprises
sample holding means which are dimensioned and configured to
releasibly grip a non-compartmentalized sample container workpiece
and injecting means for sequentially injecting distinct first and
then second reagents into the sample container workpiece at a
user-defined time interval. The apparatus further includes photon
measuring means for measuring photons emanating from the sample
container workpiece and generating signals proportional to the
measured photons. Specifically, the photon measuring means are
dimensioned and configured to sequentially generate a first
corresponding signal subsequent to injection of the first reagent
into the sample container workpiece and a second corresponding
signal subsequent to injection of the second reagent into the
sample container workpiece. Signal storage and retrieval means are
provided for storing and retrieving the first and second signals;
the signal storage and retrieval means being operationally linked
to the photon measuring means. The invention further includes
programmable control means operationally linked to and dimensioned
and configured to control overall operation of the injecting means,
the photon measuring means, and the signal storage and retrieval
means. The programmable control means are further dimensioned and
configured to perform mathematical manipulations on the first and
second signals generated by the photon measuring means. The
apparatus also includes display means to display the first and
second signals and any mathematical manipulations performed
thereon.
A second embodiment of the invention is drawn to a high-throughput
apparatus for quantifying integrated photon-generating assays.
Here, the invention is specifically designed to perform
high-throughput assays which utilize two distinct photon-generating
reactions within a non-compartmentalized sample container. Here the
apparatus comprises movable sample holding means dimensioned and
configured to releasibly grip a 96-well microtiter sample container
workpiece in which each individual well of the microtiter plate is
non-compartmentalized and one or more cooperating pairs of first
and second injecting means for sequentially injecting distinct
first and then second reagents, respectively, into an individual
well of the sample container workpiece at a user-defined time
interval. The apparatus further includes one or more corresponding
photon measuring means for measuring photons emanating from the
individual wells of the sample container workpiece and generating
signals proportional to the measured photons. The photon measuring
means are dimensioned and configured to sequentially generate a
first corresponding signal subsequent to injection of the first
reagent into the sample container workpiece and a second
corresponding signal subsequent to injection of the second reagent
into the sample container workpiece. Signal storage and retrieval
means are provided for storing and retrieving the first and second
signals; the signal storage and retrieval means being operationally
linked to the photon measuring means. Programmable control means
are operationally linked to and dimensioned and configured to
control movement and operation of the sample holding means, the
injecting means, the photon measuring means, and the signal storage
and retrieval means. The programmable control means are further
dimensioned and configured to perform mathematical manipulations on
the first and second signals generated by the photon measuring
means. Display means are provided to display the first and second
signals and any mathematical manipulations performed thereon. This
embodiment of the present invention is specifically designed to
perform one or more dual reporter, photon-generating reactions
within a 96-well microtiter workpiece.
As in the preceding embodiments, a third embodiment of the present
invention is directed to an apparatus for quantifying integrated
photon-generating assays which utilize two distinct
photon-generating reactions within a non-compartmentalized sample
container. The apparatus comprises sample holding means which are
dimensioned and configured to releasibly grip a
non-compartmentalized sample container workpiece, as well as first
injecting means for injecting a first reagent into the sample
container workpiece. Photon measuring means are provided to measure
photons emanating from the sample container workpiece at a
user-defined time interval subsequent to injection of the first
reagent and for generating a signal proportional to the measured
photons. The apparatus further includes second injecting means for
injecting a second reagent into the sample container workpiece at a
user-defined time interval subsequent to injection of the first
reagent. Photon measuring means for measuring photons emanating
from the sample container workpiece at a user-defined time interval
subsequent to injection of the second reagent and for generating a
signal proportional to the measured photons are also provided.
Here, it is preferred that the photon measuring means for measuring
photons emanating from the sample container workpiece at a
user-defined time interval subsequent to injection of the first
reagent and the photon measuring means for measuring photons
emanating from the sample container workpiece at a user-defineed
time interval subsequent to injection of the second reagent are one
in the same. Signal storage and retrieval means are included for
storing and retrieving the first and second signals; the signal
storage and retrieval means being operationally linked to the
photon measuring means. This embodiment of the invention also
includes programmable control means which are operationally linked
to and dimensioned and configured to control operation of the first
and second injecting means, the photon measuring means, and the
signal storage and retrieval means. The programmable control means
are further dimensioned and configured to perform mathematical
manipulations on the first and second signals generated by the
photon measuring means. Display means are provided to display the
first and second signals and any mathematical manipulations
performed thereon.
In all of the embodiments described above, it is preferred that the
injecting means includes an outflow orifice disposed adjacent to
the sample container workpiece, and that the outflow orifice, the
sample holding means, and the photon measuring means are disposed
within a light-tight enclosure.
A principal aim and object of the present invention is to provide
an automatic luminometer apparatus dimensioned and configured to
allow the sequential quantification of two separate and distinct
luminescent reactions within a single, non-compartmentalized sample
container.
Another aim of the present invention is to provide an automatic
luminometer apparatus dimensioned and configured to allow automatic
and high-throughput analysis of dual-reporter luminescent assays
contained within conventional 96-well microtiter plates.
Yet another aim of the present invention is to provide an automatic
luminometer apparatus dimensioned and configured to automatically
perform dual-luminescent reporter assays which employ two distinct
luciferase luminescent systems within each sample.
Still another aim of the present invention is to provide an
automatic luminometer apparatus which is dimensioned and configured
to accommodate a wide variety of sample containers, including
single-sample containers and multi-well sample containers.
A further aim of the present invention is to provide an automatic
luminometer apparatus dimensioned and configured to allow
post-acquisition manipulation of data to yield meaningful
information about the samples assayed.
A still further aim of the present invention is to provide an
automatic and programmable luminometer apparatus dimensioned and
configured to allow the sequential quantification of two separate
and distinct luminescent reactions within a single,
non-compartmentalized sample container.
Yet another aim of the present invention is to provide an automatic
and programmable luminometer apparatus dimensioned and configured
to allow automatic and high-throughput analysis of dual-reporter
luminescent assays contained within conventional 96-well microtiter
plates.
In many reporter applications, particularly genetic reporter
applications, two distinct reporters are needed to yield valid or
precise analytical data. In the prior art of enzymatic reporters,
these reporters are assayed by separate and independent
measurements of enzymatic activity. The results of these separate
assays are then combined into a common analysis. This analytical
method requiring two enzymatic reporters can be improved by
combining both measurements of enzymatic activities into a single
integrated assay process, and further improved by performing the
method on a device capable of carrying out the integrated assay
process and the subsequent data analysis. Such an analytical method
is possible using two enzymatic reporters capable of generating
luminescence in their respective assay of enzymatic activity.
The present invention provides an apparatus to perform such
dual-luminescence assays. The invention relates to luminescence
assays which include at least one reagent which rapidly quenches a
given luminescence reaction, and simultaneously initiates a second
luminescence reaction within the same sample container. Preferably,
the second luminescence reaction is initiated without any extended
incubation or delay between the two luminescent reactions. The
present apparatus is capable of measuring the first luminescent
signal, injecting quench reagents and activate reagents into the
sample, and separately measuring the second luminescent signal. The
present invention allows such dual-luminescence assays to be
performed with minimal operator input, using any type of sample
container, including 96-well microtiter plates.
The present invention relates to an automated luminometer apparatus
which is capable of acquiring, storing, and manipulating signals
generated from two distinct luminescent reactions from within a
single sample contained in a single, non-compartmentalized vessel.
By non-compartmentalized it is meant that the vessel in which the
reaction is initiated and measured defines a single, undivided, and
contiguous volume of space, such as a test tube, or a single well
of a multi-well plate. The luminescent measurements are taken
sequentially, with the first luminescent signal being measured and
quenched prior to, or simultaneous with, the initiation of the
second luminescent signal. The device is automated and includes
movable means for measuring and quantitating luminescent energy,
movable means for injecting reagents into single or multi-well
sample containers, means to manipulate the sample container(s),
optional programmable input means for programming the apparatus,
data storage, output, and display means for compiling the
information gathered by the luminescent measuring means and
presenting the information in a meaningful and/or convenient
fashion, and master control means for coordinating the various
functions of the means described above.
The preferred type of assay to be performed by the apparatus is an
integrated dual-enzyme luminescent assay which utilizes one or more
reagents to rapidly and efficiently quench enzyme-mediated
luminescent reactions. Two distinct luminescence reactions are
sequentially initiated and measured within each sample tested. The
distinct nature of the two luminescent reactions allows them to be
initiated and quantified within a single, non-compartmentalized
reaction vessel, without interference between the two reactions.
This allows two separate and distinct measurements to be taken from
a single sample without the need to subdivide the sample into two
or more portions for testing.
For sake of brevity and clarity, the present specification shall
concentrate solely on enzyme-mediated luminescent reactions, and
particularly luciferase-mediated reactions. However, the presently
disclosed invention functions equally well in assays utilizing
other, non-enzymatic, photon-generating reactions, including
phosphorescent, fluorescent, and chemiluminescent assays. The
following discussion is not limiting in any fashion, and all
functional or structural descriptions of the disclosed apparatus
applied solely to enzyme-mediated, or luciferase-mediated, assays
applies with equal force to the other non-enzyme-mediated
luminescent assays described immediately above.
An enzyme-mediated luminescence reaction is any chemical reaction
which yields photons as a consequence of the reaction, and uses an
enzyme to effectively enable the reaction. Examples include
luciferases isolated from a variety of luminous organisms, such as
the firefly luciferase of Photinus pyralis or the Renilla
luciferase of Renilla reniformis.
Luciferases are organized into groups based on the commonality of
their luminescent reactions. Generally, the luciferases within a
given group are derived from related luminous organisms, and
catalyze the same chemical reaction. For instance, beetle
luciferases, all catalyze ATP-mediated oxidation of beetle
luciferin. In contrast, anthozoan luciferases catalyze oxidation of
coelenterazine. Other enzymes in addition to luciferases mediate
luminescent reactions using both natural and synthetic substrates.
A commonly used example of this type of luminescent reaction is the
reaction of luminol with horseradish peroxidase. Another example
would be alkaline phosphatase which catalyzes a reaction with
adamantyl 1,2-dioxetane phosphate.
The present apparatus is specifically designed to enable the
quantification of two distinct luciferase-mediated reactions which
are initiated sequentially within a single sample container. In
short, the present apparatus includes means for initiating a first
luminescent reaction, means for quantifying the luminescent energy
generated by the first luminescent reaction, means for quenching
the first luminescent reaction, means for initiating a second and
distinct luminescent reaction, means for quantifying the
luminescent energy produced in the second luminescent reaction, and
means for outputting the data so gathered in various formats which
provide useful information to the user.
It is preferred that the above means be dimensioned and configured
to accommodate multiple sequential assays within conventional
96-well microtiter plates. Such microtiter plates can be either
clear or opaque.
A unique advantage of the present apparatus is that it allows two
distinct luminescent reactions to be quantified within a single
sample without the need to either divide the sample into two
portions for testing, or the need to use sub-divided sample
containers. This is extremely beneficial for testing done on small
samples. It is also cost-effective because standard sample
containers are used, rather than specialized equipment designed
specifically for the disclosed luminometer.
The present apparatus is also automatic and optionally programmable
to achieve high-throughput testing of samples contained in
multi-well microtiter plates with minimum user input.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a three-dimensional graph showing prior art signal
cross-talk radiating from a single luminescent sample within a
clear plastic, 96-well microplate.
FIG. 2 is a three-dimensional graph showing the prior art
cumulative nature of signal cross-talk radiating from multiple
luminescent samples within a clear plastic, 96-well microplate.
FIG. 3 is a schematic diagram of a first embodiment according to
the present invention.
FIG. 4 is a flow chart showing operation of a first embodiment of
the presently described invention.
FIG. 5 is a schematic diagram of a second embodiment of the present
invention including a computer controller, a keyboard to input
programming data, and output and storage means.
FIG. 6 is a flow chart showing operation of a second embodiment of
the presently described invention.
FIG. 7 shows the results of a dual-luciferase reporter assay
utilizing firefly luciferase as the first reporter, and Renilla
(sea pansy) luciferase as the second reporter as measured using the
present invention.
FIG. 8 shows the results of a dual-luciferase reporter assay
utilizing click beetle luciferase as the first reporter, and
Renilla luciferase as the second reporter as measured using the
present invention.
FIG. 9 shows the results of an analysis for Tat using a
dual-reporter luciferase assay measured using the presently
disclosed invention.
DETAILED DESCRIPTION OF THE INVENTION
Of particular interest in the present invention is a luminometer
apparatus which utilizes novel "quench-and-activate" reagents for
performing dual-reporter luminescent assays. Quench-and-activate
reagents are novel assay reagents which selectively extinguish one
luminescent reporter enzyme while simultaneously initiating another
distinct enzyme-mediated luminescence reaction. The preferred
protocol is to initiate and measure the second luminescent reaction
promptly after the first luminescent reaction is extinguished,
without any extended delay or incubation period. Preferably, the
second luminescent reaction is initiated within one minute after
the first luminescent reaction is extinguished. This assay allows
for the sequential measurement of two separate and distinct
luminescent reporters within one sample. The presently described
apparatus is a device for automatically performing a dual-reporter
luminescent assay.
A great benefit of the dual-reporter assay is that one of the
luminescent reporters can be used as an internal standard, while
the other luminescent reporter is used to report the impact of the
experimental variables. In operation, one of the two
enzyme-mediated luminescence reactions is first initiated by
addition of a first initiating reagent or reagents into the
experimental system. The luminescence signal produced by the first
enzymatic reaction is then measured. The first enzymatic reaction
is then specifically and selectively quenched by adding a
quench-and-activate reagent which simultaneously quenches the first
enzymatic reaction and initiates a second enzymatic reaction. The
quench and activate reagent may be a single mixture of components
(as in a solution), or may be two separate mixtures added to the
reaction by separate means. The luminescent signal produced by the
second enzymatic reaction is then measured in the same fashion as
the first reaction. The information is stored in retrievable
storage means, preferably magnetic storage media, and output to an
output device, such as a CRT screen.
As a research tool for molecular biologists, luminescent enzyme
reporter systems are more sensitive than the other, more
traditional, enzyme reporter systems. Luciferase assays, in
particular, are also much more rapid than those of other enzyme
reporter systems. These features make luminescence-generating
enzymes the preferred reporters of genetic and physiological
activity.
A first embodiment of the present invention is shown in schematic
form in FIG. 3. Reference 10 designates a photon-detector which may
be static or movable. If movable, the photon-detector can be
translated in both the vertical and horizontal axes to allow easy
access and maintenance of the entire apparatus. This movement is
accomplished by conventional mechanical, electromechanical, or
magnetic means, such as servomotors, solenoids, etc. The
photon-detector can be of any conventional design, including a
single or compound photomultiplier tube, a charge-coupled device
(CCD), or similar device capable of accurately measuring
luminescent energy.
The photon-detector senses the energy of a luminescent reaction and
produces output signals corresponding to the intensity of the
luminescence measured. A sample cuvette 11 is placed within a
sample holder 12, the sample holder 12 being situated in
operational relationship to the photon-detector 10. The sample
holder 12 may also be movable or static. If movable, the sample
holder is preferably translatable in either or both the vertical
and horizontal axes to allow flexible positioning of samples, and
convenient access for loading and unloading samples. The sample
cuvette 11 may be a single sample container, as shown in FIG. 3, or
a multi-well container, such as a 96-well microtiter plate.
One or more reagent injectors 14 is situated in operational
relationship to the sample holder 12 and the sample cuvette 11. An
injector output orifice, 15 is disposed adjacent to the sample
container workpiece. The reagent injector 14 is capable of
injecting liquid or solid reagents via the injector output orifice
15 into the sample cuvette 11 when the cuvette is placed within the
sample holder 12. The reagent injector can be an assembly whereby
one or more liquids are forced from one or more reservoirs through
a conduit by a peristaltic pump, a vacuum-controlled aspirator, a
reciprocating pump, and the like. For solid reagents, the reagent
injector may be a conveyor belt, screw conveyor, etc.
In terms of overall functionality, the reagent injector 14 and
injector output orifice 15 may be any type of conventional assembly
dimensioned and configured to deliver material to a given location
without limitation.
The reagent injector outflow orifice 15, sample holder, sample
cuvette, and photon-detector are enclosed within a light-tight
enclosure 18. The enclosure may comprise a suitably opaque lid or
cover which encloses the various sub-assemblies, and includes a
movable closure to allow access to the interior thereof.
The entire apparatus is controlled by a controller 20 operationally
linked to the various components of the apparatus by a plurality of
control lines 22, 24, 26, and 28. As depicted schematically in FIG.
3, a data storage and retrieval device 16 is operatively linked to
the photon-detector 10 to store data quantified by the
photon-detector. The data storage and retrieval device 16 is also
controlled by the controller 20.
The controller 20 can be any type of controller known in the art.
This includes, but is not limited to, programmable computer control
means, microprocessors, digital signal processors, LSI chips, VLSI
chips, ELSI chips, RISC processors, analog to digital converters
and signal processors, digital to analog converters and signal
processors, read only memory chips (ROM), erasable programmable
read only memory chips (EPROM), conventional electric circuitry,
conventional electronic circuitry, or mechanical means.
The control means 20 may be programmable. By "programmable," it is
meant that the control means 20 may be dimensioned and configured
to store and execute one or more pre-determined instruction sets
entered either by the end-user (end-user programmability), or
pre-determined and fixed at the time of manufacture (factory-fixed
programmability). Flexible, end-user programmability allows the
user to adapt the apparatus to perform various dual-reporter assay
protocols to fit a given need. Factory-fixed programmability
provides lower cost and ease of operation for fixed protocols.
The data storage and retrieval device 16 may be any such device
known to the industry, including, but not limited to, magnetic
storage media without limitation, including floppy disk drives,
hard disk drives, and tape drives; magneto-optical storage media
without limitation including WORM drives and erasable
magneto-optical media; optical storage media without limitation,
such as compact discs, LASER discs, and the like, random access
memory chips (RAM), or conventional electric and/or electronic
storage circuitry, or conventional mechanical storage means, such
as punch cards.
The control lines 22, 24, 26, and 28 can be conventional wire
connectors, such as coaxial cables, 10-pin connectors, SCSI
connectors, and the like, which are capable of transmitting signals
to and from the control means 20 to the various components of the
present apparatus. The control lines can also be optical fiber
cable, or wireless electromagnetic frequency control lines, such as
infrared or microwave transmitters capable of receiving and
transmitting signals to and from the control means, 20.
The invention may also include display means, such as a cathode ray
tube or printer device, means for entering programming and other
information into the controller, such as a keyboard, as well as an
output means for outputting data compiled by the data storage and
retrieval device.
A flow chart depicting the general operation of the first
embodiment of the present invention is shown in FIG. 4. The
operator of the device shown in FIG. 3 begins the assay by
optionally measuring the dark current of the photon-detector 10.
The operator may then optionally initiate the first luminescent
reaction manually (step 2). This is done within the sample cuvette
11. The sample cuvette is then placed within the sample holder 12,
and the light-tight enclosure 18 sealed to prevent ambient light
from interfering with measurement of the luminescent energy
generated by the reaction (step 3). Otherwise, the sample cuvette
is placed into the luminometer, and the first initiating reagent is
injected into the sample automatically (step 4).
The luminescence energy of the first enzyme-mediated luminescent
reaction is then measured by the photon-detector 10, and the
measured signal sent to the data storage and retrieval device as
shown by arrow number 1 of FIG. 3. (Steps 5 and 6 of FIG. 4). One
or more "quench-and-activate" reagents are then injected into
sampled cuvette 11 via the reagent injector 14 through the output
orifice 15 (step 7). As noted above, the quench-and-activate
reagent(s) quenches the first luminescent reaction prior to, or
while simultaneously initiating a second luminescent reaction. The
photon-detector 10 is then used to measure the luminescent
intensity of the second enzyme-mediated luminescent reaction. This
second signal, designated by arrow number 2, is then output to the
data storage and retrieval device 16. (Steps 8 and 9.) Optional
steps here include injecting a quench reagent into the sample, and
again measuring the dark current of the photon-detector. The first
and second signals may then be manipulated using the control means
20 to yield meaningful results, such as a ratio of the two
signals.
As noted above, the actual quantitation of the luminescent
reactions takes place within the light tight enclosure 18.
A second and preferred embodiment of the present invention is shown
in FIG. 5. Here, the photon-detector 10, reagent injector 14,
injector output orifice 15, light-tight enclosure 18, controller
20, and data storage and retrieval means 16, are the same as
described for FIG. 3. Additionally, it must be noted that there may
be a plurality of photon-detectors 10 and reagent injectors 14 in
order to maximize sample throughput. Such duplication of functional
parts can be accomplished by mounting a plurality of the reagent
injectors and photon-detectors upon a movable boom which enables
the injectors and photon-detectors to be accurately positioned in
operational relationship to multiple samples simultaneously. In
this fashion, a plurality of samples can be analyzed
simultaneously.
In the embodiment represented in FIG. 5, the sample holder 11 is a
multi-well sample holder, such as a conventional 96-well microtiter
plate. The plate 11 rests upon a movable stage 13 which is
dimensioned and configured to position the plate into operational
relationship with the photon-detector 10 and the reagent injector
output orifice 15. The movable stage may also include releasible
anchoring means for securely and releasibly fastening the
multi-well plate to the stage. Preferably, the movable stage 13 is
translatable in the horizontal plane, as indicated by the double
arrow. Additionally, the movable stage may be translatable in the
vertical axis to facilitate the initial loading and ultimate
removal of the plate 11 from the apparatus. (This also allows the
apparatus to accommodate non-standard sample holders of different
sizes.)
Also, both the photon-detector 10 and the reagent injector output
orifice 15 may be translatable to facilitate ease of operation,
access, and maintenance of the disclosed apparatus. Movement of the
stage, photon-detector, and reagent injector may be controlled by,
for instance, a worm-screw rotated by a stepping motor.
Servo-motors, hydraulic controls, pneumatic controls, and other
conventional means for controlled movement may also be employed
with equal success.
The preferred apparatus depicted in FIG. 5 also includes a data
entry device 32 and a data display device 30. The data entry device
32 is preferably a standard keyboard entry device.
The display device 30 may be a standard cathode-ray tube screen or
light-emitting or liquid crystal diode display, and/or a hard-copy
display device, such as a means for printing accumulated data. Such
devices are well known and widely used.
The control lines 22, 24, 28, 34, 36, and 38 are the same as those
described for FIG. 3.
The operation of the luminometer device described in FIG. 5 is
shown in flow-chart form in FIG. 6. Preliminarily, the multi-well
plate 11 is placed into the apparatus, releasibly anchored to the
movable stage 13, and the light-tight enclosure 18 is then sealed.
The subsequent actions taken by the apparatus are initiated by
commands entered from the data entry device 32, and, once
initiated, may be executed automatically, without further user
input, by the control means 20. The dark current of the
photon-detector 10 may then be optionally determined to establish a
base-line signal. In step 2, the reagent injector output orifice 15
is placed into operational alignment with the first well of the
multi-well plate to be tested. This is accomplished by
translational movement of the movable stage 13. This movement is
controlled by the controller 20, and may be indexed to move
accurately from well to well when multi-well plates are
utilized.
Also in step 2, the reagent injector 14 is used to inject a first
reagent into the well to initiate a first luminescent reaction. The
luminescence of the first reaction is then measure by the
photon-detector 10 in step 3. In step 4, the quantified signal is
transferred via the controller 20 to the data storage and retrieval
means 16 by the control lines 24 and 28.
In step 5, one or more "quench-and-activate" reagents are injected
into the well by reagent injector 14. As depicted in FIGS. 3 and 4,
the reagent injector includes only one reservoir. This is for
clarity purposes only. The injector means may include a plurality
of reservoirs from which to draw and inject various liquid and/or
solid reagents into the sample wells of plate 11. The injection of
the "quench-and-activate" reagent halts the first luminescent
reaction, and initiates a second and distinct luminescent reaction.
The luminescent energy of the second reaction is then quantified,
and the signal transferred to the data storage and retrieval means
16 in steps 6 and 7 of FIG. 6.
At this point in the operation of the apparatus, two optional steps
may be included: a quench reagent may be injected into the well to
quench the second luminescent reaction, followed by measurement of
the dark current of the photomultiplier 10. These steps may be
omitted, in which case the operation of the device would proceed
directly to step 10.
The entire process from steps 2 through 9 is then repeated to
acquire data for the remaining sample wells. The process is
repeated until all sample wells have been assayed. The data so
generated is accumulated in data storage registers corresponding to
the sample well tested within the data and storage retrieval means
16. In this manner, the collected data can be matched to the sample
well from which it came.
The collected data is then manipulated via the control means 20, or
by external data manipulation means (not shown), to yield useful
information which is then displayed by the display means 30.
Illustrative data manipulations would include generating ratios of
the two luminescent signals per sample; means, ranges, medians, and
the like between samples; curve-fitting calculations, such as least
squares analyses, and non-linear curve-fitting, signal-to-noise
enhancement calculations, such as Fourier transformation analysis
and related manipulations; and precision, accuracy, and threshold
detection calculations and compilations. Such data manipulation
techniques are well-known in the art.
The present luminometer apparatus may also include components not
shown which are conventionally found on luminometers. For instance,
the apparatus may include a movable shutter to block the entry of
light into the photomultiplier tube. This shutter can be open and
closed. In the closed position, the shutter allows the dark current
of the photomultiplier tube to be measured. The luminometer
apparatus may include more than one type of photo-detecting means
in order to accurately perform assays which generate light of
different wavelengths, such as U.V. light.
FIGS. 7 and 8 illustrate representative dual-reporter assays
utilizing firefly, Renilla, and click beetle luciferases. In FIG.
7, a first luminescent reaction, mediated by firefly luciferase was
initiated and quantified. The initiating reagent is designated BLA,
which is an acronym for Beetle Luciferase Activation reagent. The
photons generated by this first reaction are tabulated in the
left-hand column of FIG. 7. (appr. 100,000 Relative Light Units,
RLU).
The first luminescent reaction was then quenched and a second,
Renilla-mediated, reaction initiated by the injection of a Beetle
Luciferase Quench, Renilla Luciferase Activation reagent (BLQRLA
reagent). The results of this measurement are depicted in the
right-hand column of FIG. 7. The center column of FIG. 7 depicts
the residual activity of the first luminescent reaction when a
quench reagent only is added to the sample after the initiation of
the first luminescent reaction.
An analogous experiment is depicted in FIG. 8. Here, click beetle
luciferase was used as the first luminescent reporter, while
Renilla luciferase was used as the second luminescent reporter. The
left-hand column shows initiation and quantification of the click
beetle luciferase reporter. Residual activity after addition of a
quench reagent only is shown in the middle column. The right-hand
column shows quenching of the first luminescent reaction, and
activation of a second, distinct, luminescent reaction
(Renilla-mediated).
FIG. 9 depicts the results of a dual-luminescent assay for the Tat
protein. An experimental genetic vector containing the firefly
luciferase gene (luc) coupled to the genetic promoter of the Human
Immunodeficiency Virus (HIV LTR) was introduced into a human cell
line designated 293. In this experiment, the activity of the HIV
LTR is expected to be activated by the Tat protein, a gene product
generated during HIV replication. Also introduced as an
experimental control was a genetic vector containing the Renilla
luciferase gene coupled to the SV40 promoter. The SV40 promoter is
expected to be unaffected by the Tat protein. The inclusion of the
control vector provides an internal control for variability in the
process of introducing the genetic vectors into the cells and in
cell growth.
Cells containing both vectors were grown in the presence and
absence of Tat in the growth medium, and subsequently lysates of
the cells were made. Using the dual-luciferase reporter method,
both reporters were measured from individual lysate samples.
"Reporter Response" is the relative amount of luminescence measured
from each lysate, and the "Normalized Response" is the ratio of the
firefly luciferase assay divided by the Renilla luciferase assay.
Comparison of these normalized responses indicates the amount of
activation (or "Induction") caused by the Tat protein. The ratio of
the normalized responses is the induction ratio.
The results also show that measurement of the first reporter does
not affect the second reporter. This is true since the first
reporter increases by more than 10-fold without any detectable
increase in the second reporter (in fact, the second reporter is
somewhat reduced, a consequence of the experimental variability
described above). This is especially impressive since the first
reporter yields roughly 1000-fold more luminescence than the second
reporter in this particular experiment. Thus, the light from the
first reporter must be quenched roughly more than 100,000-fold
before measurement of the second reporter. This is as expected from
the other figures (FIGS. 7 and 8) showing the quenching capacity of
the quench-and-activate reagent.
The presently disclosed invention is not limited to the exact
descriptions included above, but includes all equivalent
embodiments thereof, accomplished by equivalent means now known, or
equivalent discovered in the future.
BIBLIOGRAPHY
Blaise, C., et al. (1994) BioTechniques: 16, 932-937.
Bronstein, I., et al. (1994) Anal-Biochem.: 219, 169-181.
Denburg, et al. (1969) Archives of Biochemistry and Biophysics:
134, 381-394.
Denburg, J.L., and McElroy, W.D. (1970) Archives of Biochemistry
and Biophysics: 141, 668-675.
Flanagan, W. M. et al. (1991) J. Virology: 65, 769-786.
Jain, V. K. and Magrath, I. T. (1992) BioTechniques: 12,
681-683.
Kondepudi, T., et al., Poster abstract #725, presented at annual
meeting of the American Society of Cell Biology, Dec. 10-14, 1994,
San Francisco, Calif.
Leckie, F. et al. (1994) BioTechniques: 17, 52-57.
Lee, et al. (1970) Archives of Biochemistry and Biophysics: 141,
38-52.
Thompson, J. F., et al. (1991) Gene: 103, 171-177.
U.S. Pat. No. 3,756,920 to Kelbaugh et al., issued Sep. 4,
1973.
U.S. Pat. No. 4,235,961 to A.T. Lundin, issued Nov. 25, 1980.
U.S. Pat. No. 4,390,274 to Berthold et al., issued Jun. 28,
1983.
U.S. Pat. No. 4,459,265 to Berglund, issued Jul. 10, 1984.
U.S. Pat. No. 4,755,055 to Johnson et al., issued Jul. 5, 1988.
U.S. Pat. No. 4,818,883 to Anderson et al., issued Apr. 4,
1989.
U.S. Pat. No. 5,035,866 to Wannlund, issued Jul. 30, 1991.
U.S. Pat. No. 5,043,141 to Wilson et al., issued Aug. 27, 1991.
U.S. Pat. No. 5,082,628 to Andreotti et al., issued Jan. 21,
1992.
U.S. Pat. No. 5,139,745 to Barr et al., issued Aug. 18, 1992.
U.S. Pat. No. 5,159,197 to Wannlund, issued Oct. 27, 1992.
U.S. Pat. No. 5,283,179, to Wood, issued Feb. 1, 1994
U.S. Pat. No. 5,290,708 to Ashihara et al., issued Mar. 1,
1994.
U.S. Pat. No. 5,316,726 to Babson et al., issued May 31, 1994.
U.S. Pat. No. 5,380,487 to Choperena et al., issued Jan. 10,
1995.
European Patent Application 0 025 350 to Holley, published Mar.
1981.
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